Therapeutic protein compositions and methods

ABSTRACT

Compositions of the inventive concept provide a therapeutic protein with less than 2% contamination by the therapeutic protein in denatured form. Such compositions provide enhanced specific activity and improved stability on storage and/or in serum than corresponding therapeutic protein preparations resulting from conventional isolation methods.

This application is a continuation of U.S. patent application Ser. No.16/740,286, filed Jan. 10, 2020, which claims the benefit of U.S.Provisional Patent Application No. 62/903,659 filed on Sep. 20, 2019.These and all other referenced extrinsic materials are incorporatedherein by reference in their entirety. Where a definition or use of aterm in a reference that is incorporated by reference is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is therapeutic proteins, specificallytherapeutic proteins isolated from blood or blood products.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

A variety of proteins are currently isolated from human blood productsfor therapeutic application. Examples of such proteins include albumin,immunoglobulins, alpha-1 antitrypsin, can various clotting factors.These therapeutic proteins are often used in large amounts and highconcentrations. Albumin is used at high concentrations (up to 25% byweight) to restore blood volume in surgical, trauma, and burn patients.Immunoglobulin preparations (which typically contain about 10% by weightIgG) are used as immunomodulating agents to treat a wide range ofdiseases and conditions, and in addition to providing support for aweakened immune response can act by modulating complement activity,suppressing idiotypic antibodies, saturating macrophage Fc receptors,and suppressing of a variety of inflammatory mediators. Weekly infusionwith alpha-1 antitrypsin (at about 60 mg/kg body weight) is used totreat or slow the progression of genetic disease associated with alpha-1antitrypsin deficiency. Clotting factors (such as factor VIII, factorIX, von Willebrand factor, and other components of the clotting cascade)find use in treating hemophilia, and are provided in concentrated formin order to permit treatment by simple injection.

Due to the quantities required such therapeutic proteins are isolatedfrom blood products (such as plasma) using scalable processes, typicallya series of steps that can include precipitation, filtration,resolubilization, and treatment with chromatography media. The highdegree of purity required generally necessitates the use of multipleseparation steps. Typically, Cohn fractionation of serum is used, whichinvolves a series of precipitation steps resulting from the stepwiseaddition of alcohol. Such extensive processing, however, can result inthe accumulation of denatured forms of the therapeutic protein in thepurified fractions. Such denaturation can lead to adverse reactions uponadministration, reduced activity, reduced half-life on storage(particularly in liquid formulations), and/or reduced serum half-lifefollowing administration.

Thus, there is still a need for therapeutic protein preparation withminimal contamination and little to no denatured protein content.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions including atherapeutic protein that have been processed in such a manner as to havea low content of denatured therapeutic protein and/or othercontaminants. Such compositions provide high specific activity and/orstability relative to corresponding preparation of therapeutic proteingenerated by conventional processes.

Compositions of the inventive concept include a therapeutic proteinisolated from blood product (such as a modified cryo-poor plasma), wherethe therapeutic protein is present as a native form and a denaturedform, and where the denatured form provides from about 0.01% to about1%, 2%, 3%, 4%, or 5% of either the total amount of therapeutic proteinor the native form of the therapeutic protein. In some embodiments thetherapeutic protein is exposed to a precipitant during isolation. Insome embodiments the therapeutic protein is alpha-1 antitrypsin, and thecomposition is greater than 90% inhibitory when tested with active-sitetitrated porcine pancreatic trypsin usingN-benzoyl-L-arginine-para-nitroanilide hydrochloride (L-BAPNA) assubstrate. In some embodiments the therapeutic protein is that of animmunoglobulin, and has an in vivo half-life following administrationthat is at least 5%, 10%, 15%, 20%, or 25% greater than animmunoglobulin fraction prepared using Cohn fractionation. In someembodiments the therapeutic protein is albumin colorless at aconcentration of at least 4%, 5%, 8%, 10%, 15%, 20%, or 25%.

Another embodiment of the inventive concept is method of preparing atherapeutic protein composition as described above, by thawing frozenplasma at a temperature of from about 1° C. to about 6° C. in thepresence of a precipitant to generate a modified cryoprecipitate and amodified cryo-poor plasma, separating the modified cryoprecipitate fromthe modified cryo-poor plasma, applying the modified cryo-poor plasma toa chromatography media without an intervening precipitation orsignificant dilution step to produce an unbound fraction and a boundfraction, and recovering a therapeutic protein that has only from about0.01% to about 5% denatured therapeutic protein from either the unboundfraction or the bound fraction. Suitable precipitants include organicacids, salts of organic acids (such as sodium citrate), inorganic salts,and hydrophilic polymers.

Another embodiment of the inventive concept is a method of preparing atherapeutic protein composition as described above by adding anonvolatile precipitant to a blood product to provide a precipitantconcentration that does not result in the formation of a precipitate toform an intermediate solution, removing water from the intermediatesolution while retaining the precipitant until a target precipitatingconcentration of the precipitant is reached to generate a precipitateand a supernatant, separating the precipitate from the supernatant, andrecovering a therapeutic protein that includes from about 0.01% to about1% denatured therapeutic protein from either the supernatant or theprecipitate. Suitable precipitants include organic acids, salts oforganic acids (such as sodium citrate), inorganic salts, and hydrophilicpolymers. Water can be removed by evaporation (e.g. under reducedpressure) or by ultrafiltration. In some embodiments the therapeuticprotein is recovered from the supernatant. In some embodiments thetherapeutic protein is recovered from the precipitate.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Embodiments of the inventive concept provide therapeutic proteincompositions with a low content of denatured therapeutic protein andother contaminants. The resulting composition provides the therapeuticproteins in a state that more closely resembles their state in vivo(e.g. prior to being subjected to purification techniques). Suchtherapeutic protein preparations demonstrate higher specific activities,extended stability, and/or prolonged in vivo half life followingadministration when compared to corresponding preparations resultingfrom prior art methods.

This is achieved, at least in part, by utilizing preparative methodsthat scrupulously avoid protein denaturation. Such methods, for example,can eliminate the use of denaturing organic solvents (e.g. alcohols,ketones, etc.), utilize process steps that minimize the localconcentration of precipitating agents, use careful mixing to avoid theproduction of foam or froth, and/or avoiding or minimizing the use ofdenaturing precipitants (such as organic solvents). Similarly, suchprocess steps can avoid extremes of pH (for example, maintaining a pHbetween about 6.5 and about 8.5, or a pH from about 6.8 to about 7.5)and/or extremes of temperature (e.g. maintaining a temperature of about4° C. to 8° C., or a temperature of about 18° C. to about 25° C.throughout processing). Nonexclusive examples of such methods areprovided below.

The source material for the vast majority of blood products is frozenplasma obtained from commercial collection centers. Slowly thawing thismaterial at low temperature (typically from 1 to 6° C.) generates anintermediate blood product that contains precipitated proteins (i.e.cryoprecipitate or “cryo”) and a protein-rich supernatant (cryo-poorplasma). Cryoprecipitate includes some of the fibrinogen content of thesource plasma, as well as clotting factors and fibrin. Cryo-poor plasmais rich in cold-soluble proteins and is frequently used as a source ofpharmaceutical proteins.

Cryo-poor plasma can have a protein content and/or denatured proteincontent that renders it unsuitable for direct application toconventional chromatographic separations without an intervening dilutionor processing step. Surprisingly, the Inventors have found that theinclusion of a low concentration of a precipitant (i.e. a concentrationthat does not result in observable precipitation when applied to serumand/or plasma) in the thawing process can alter the protein distributionbetween cold-soluble and cold-insoluble fractions in the resultingpreparation. The resulting modified cryo-poor plasma has been found tohave a protein content that permits direct application to chromatographymedia (e.g. size exclusion media, ion exchange media, hydrophobicinteraction media, affinity media, mixed-mode chromatography media,etc.) without intervening dilution and/or precipitation steps. Thisadvantageously minimizes process steps, each of which provides unwantedopportunities for denaturation. Such chromatography steps can beperformed while maintaining a low temperature (e.g. from 4° C. to 8° C.)similar or identical to that used in the initial thawing step, in orderto reduce the chance of protein denaturation. Similarly, pH can be heldconstant or controlled within a restricted range (e.g. pH 6 to pH 8, pH6.5 to 7.8, pH 6.8 to 7.2) during these steps in order to minimizedenaturation.

In a preferred embodiment of the inventive concept the chromatographymedia is an affinity media. This advantageously both simplifies andreduces the time and materials required for plasma processing time. Inaddition, reduction in the number of processing steps can reduce thedegree to which sensitive protein species are denatured, resulting inimproved stability on storage, improved in vivo half life followingadministration, and/or improved specific activity.

The inventive concept also provides compositions and methods in which anon-volatile precipitant (e.g. a sulfate salt, a phosphate salt, a saltof an organic acid, and or a soluble polymer) is introduced to a bloodproduct containing one or more target proteins and one or morecontaminating proteins. In some embodiments the precipitant is providedin an amount or a concentration that does not result in the formation ofa visible precipitate. Water is then removed from the resulting reactionmixture to increase the concentration of both protein and precipitantsimultaneously. When the protein concentration and precipitantconcentration reach the desired target values a protein precipitateforms, and is subsequently separated from the supernatant fraction.Mixing with the precipitant and separation of precipitate andsupernatant fractions can be performed at a constant temperature orwithin a restricted temperature range (e.g. 15° C. to 25° C., 18° C. to22° C.) in order to reduce denaturation. Similarly, pH can be heldconstant or controlled within a restricted range (e.g. pH 6 to pH 8, pH6.5 to 7.8, pH 6.8 to 7.2) during these steps in order to minimizedenaturation.

Depending on the nature of the target protein and the precipitant thetarget protein can be present in the precipitate or in the supernatantfraction. Since the protein concentration is increased as theprecipitant concentration increases during this process the distributionof proteins between the precipitate and supernatant fractions isdifferent and distinct from that produced in conventional precipitationprocesses in which protein concentration is decreased or at bestmaintained as precipitant concentration increases. In some embodiments asupernatant obtained from such a precipitation process can be subjectedto additional solvent (i.e. water) removal to further increase proteinand precipitant concentration and generate a second precipitate andsecond supernatant fraction.

Simultaneously increasing protein concentration while increasingprecipitant concentration in this fashion increases the efficiency ofprecipitation, providing for increased yields of target proteins. Inaddition, initial introduction of the precipitant at concentrations thatdo not yield a visible precipitate precludes the formation of unwantedprotein precipitants due to localized high concentrations of precipitant(as found on precipitant addition in conventional processes), decreasingthe chance of undesirable protein denaturation and improving thespecific activity of target proteins recovered from such processes.

As noted above, in embodiments of the inventive concept precipitants areselected to be nonvolatile (i.e. having a higher vapor pressure thanwater of the aqueous solution of protein under the current ambientcondition). The amount of precipitant used can vary depending upon thenature of the precipitant. Suitable precipitants are preferablynondenaturing, and can include organic acids and salts of organic acids(e.g. sodium citrate), inorganic salts (e.g. ammonium sulfate, sodiumsulfate, sodium chloride), and hydrophilic polymers (e.g. PEG, dextran,etc.). For example, if an organic salt such as sodium citrate is used itcan be provided at concentrations ranging from about 0.1%, 0.2%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, or less than about20% (w/v). Similarly, if an inorganic salt such as ammonium sulfate isused it can be provided at concentrations ranging from about 0.1%, 0.2%,0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, or less thanabout 20% (w/v). If a hydrophilic polymer such as PEG is used it can beprovided at concentrations ranging from about 0.01%, 0.02%, 0.05%, 0.1%,0.2%, 0.5%, 0.7%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 7%, or less than about10% (w/v).

Water can be removed from an aqueous solution of proteins containing aprecipitant by any suitable method. The method used can depend on thenature of the precipitant. For example, if the precipitant is ahydrophilic polymer filtration (e.g. ultrafiltration, diafiltration)utilizing a membrane with a molecular weight cutoff that is less thanthat of the molecular weight of the hydrophilic polymer can be used toremove water from the aqueous solution. In another embodiment,evaporation (at ambient pressure or at reduced pressure) can be used toremove water from the aqueous solution.

In a preferred embodiment of the inventive concept the therapeuticprotein so obtained as a result of treatment of a blood product.Suitable blood products include serum plasma, cryo-poor plasma, amodified cryo-poor plasma as described above, resolubilizedcryoprecipitate, and modified resolubilized cryoprecipitate as describedabove. In some embodiments the blood product can have been throughvarious process steps (e.g. dilution, pH adjustment, addition ofprotease inhibitors, addition of anticoagulants, precipitation, etc.)prior to being introduced to a method of the inventive concept.

In some embodiments of the inventive concept, a supernatant or aprecipitate generated by a method of the inventive concept can befurther processed to recover one or more target proteins and/or removeundesirable contaminants. In such embodiments a precipitate generated bythe method can be redissolved prior to the additional processing.Suitable additional processing steps include further water removal froma supernatant, conventional precipitation by addition of precipitatingamounts of a precipitant, and/or chromatography (e.g. using ionexchange, hydrophobic interaction, affinity, mixed-mode, and or sizeexclusion chromatography media).

Chromatography media utilized in such additional processing can have anysuitable formulation and configuration. Suitable media can be formulatedfor size exclusion, ion exchange, hydrophobic interaction, affinity,and/or mixed mode separations. Suitable media can be provided as porousgranules or beads, non-porous granules or beads, filters, fibers, and/orporous membranes. Structural portions of chromatography media can bebased on any suitable materials. Examples include but are not limited topolysaccharides (such as cross-lined dextran), synthetic polymers,and/or inorganic materials (such as hydroxyapatite). Chromatographymedia can be provided in any suitable geometry. Suitable geometriesinclude open or sealed chromatography columns, radial chromatographycolumns, cartridges, membrane housings, etc.

In an example of a method of the inventive concept, a blood product(such as plasma) is obtained and blended with an equivalent volume of 8%(w/v) sodium citrate with rapid stirring, to form an aqueous proteinsolution with a non-precipitating sodium citrate concentration of 4%(w/v). The aqueous protein solution is transferred to a sealed containerand the air pressure reduced to below that of the vapor pressure ofwater at the ambient temperature, resulting in the rapid evaporation ofwater from the solution. In some embodiments a small amount of air iscontinually bled into the sealed container during this process toprevent equilibration of water vapor within the sealed chamber. Water isremoved until the volume of the aqueous solution is reduced to bring thesodium citrate concentration to between about 10% and 12% whileincreasing the protein concentration, resulting in the formation of avisible precipitate. The precipitate can then be separated from thesupernatant fraction, for example by filtration or by centrifugation.Such separation can be performed at ambient or reduced pressure.

In another example of a method of the inventive concept a blood product(such as plasma) is obtained and blended with an equivalent volume of 2%(w/v) polyethylene glycol (PEG) having a mean molecular weight of 5 kDusing rapid stirring, to form an aqueous protein solution with anon-precipitating PEG concentration of 1% (w/v). The aqueous proteinsolution is subjected to ultrafiltration using a non-fouling membranehaving a 3 kD molecular weight cutoff, resulting in the rapid removal ofwater and other low molecular weight species from the solution whileretaining the precipitant. Ultrafiltration is continued until the volumeof the aqueous solution is reduced to about 25% of the original volumeof the aqueous protein solution, bringing the PEG concentration to about4% w/v while increasing the protein concentration and resulting in theformation of a visible precipitate. The precipitate can then beseparated from the supernatant fraction, for example by filtration or bycentrifugation.

A variety of pharmaceutically useful proteins can be obtained frommethods of the inventive concept at high yield, specific activity,purity, in vitro stability, and/or in vivo stability. Such proteinsinclude fibrinogen, factor VII, factor VIII, factor IX, factor XIII, vonWillebrand factor, fibronectin, immunoglobulins, alpha-1 antitrypsin,protein C, protein S, C1 esterase inhibitor, antithrombin 3, thrombin,and/or albumin.

As noted above, therapeutic proteins found in compositions of theinventive concept have a low content of denatured therapeutic protein.The portion of therapeutic protein that is in a denatured statefollowing application of a method of the inventive concept to a suitablestarting material can range from about 0.01% to about 1%, 2%, 3%, 4%,5%, 8%, or 10% of the total therapeutic protein content or of the nativetherapeutic protein content of the mixture. The therapeutic proteins soproduced can have a high degree of storage stability and/or stability invivo following administration and/or can have high specific activity,relative to corresponding therapeutic protein preparation made usingconventional methods (e.g. Cohn fractionation). For example,immunoglobulin preparation of the inventive concept can havesignificantly greater (e.g. at least 10% greater) shelf life and/or invivo half life following administration than correspondingimmunoglobulin preparations manufactured by conventional Cohnfractionation (i.e. ethanol precipitation). Similarly, alpha-1antitrypsin preparations of the inventive concept can be greater than90% inhibitory when tested with active-site titrated porcine pancreatictrypsin using N-benzoyl-L-arginine-para-nitroanilide hydrochloride(L-BAPNA) as a substrate.

Similarly, contaminants (e.g. bilirubin, fatty acids, etc.) can beremoved to a very high degree while retaining from 90% to 99.99% of thetherapeutic protein in active, native conformation, yielding proteinsolutions that are colorless or essentially colorless at high proteinconcentrations (e.g. 10 mgmL⁻¹ or greater). This can be particularlyuseful for albumin, as such a reduction in contaminants can result in aconcomitantly increased capacity for adsorption of undesirablemolecules, such as circulating amyloid plaque materials and/or smallorganic molecules (e.g. therapeutic drugs, drugs of abuse, drugmetabolites, etc.) by this protein when applied therapeutically.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A composition consisting of an isolatedtherapeutic protein composition comprising alpha-1-antitrypsin and aprecipitant, wherein the isolated therapeutic protein compositioncomprises non-denatured alpha-1-antitrypsin in a first amount anddenatured alpha-1-antitrypsin in a second amount, wherein the isolatedtherapeutic protein composition is a product of a treatment with theprecipitant, wherein the second amount is from 0.01% to 5% of the firstamount, and wherein the therapeutic protein composition is isolated froma blood product, and wherein the denatured alpha-1-antitrypsin hasreduced specific activity, and wherein the composition is greater than90% inhibitory when tested with active-site titrated porcine pancreatictrypsin using N-benzoyl-L-arginine-para-nitroanilide hydrochloride(L-BAPNA) as a substrate.
 2. The composition of claim 1, wherein theblood product is a modified cryo-poor plasma.
 3. A compositionconsisting of an isolated therapeutic protein composition comprising animmunoglobulin and a precipitant, wherein the isolated therapeuticprotein composition comprises a non-denatured immunoglobulin in a firstamount and a denatured immunoglobulin in a second amount, wherein theisolated therapeutic protein composition is a product of a treatmentwith the precipitant, wherein the second amount is from 0.01% to 5% ofthe first amount, and wherein the isolated therapeutic proteincomposition is isolated from a blood product, and wherein the denaturedimmunoglobulin is denatured to an extent that reduces its in vivohalf-life, and wherein the isolated therapeutic protein composition hasan in vivo half-life that is at least 10% greater than an immunoglobulinfraction prepared using Cohn fractionation.
 4. A composition consistingof an isolated therapeutic protein composition comprising albumin and aprecipitant, wherein the isolated therapeutic protein compositioncomprises a non-denatured albumin in a first amount and a denaturedalbumin in a second amount, wherein the isolated therapeutic proteincomposition is a product of a treatment with the precipitant, whereinthe second amount is from 0.01% to 5% of the first amount, and whereinthe isolated therapeutic protein composition is isolated from a bloodproduct, and wherein the denatured albumin is denatured to an extentthat can induce discoloration, and wherein the composition is colorlesswhen the isolated therapeutic protein composition is provided in asolution at a concentration of at least 10% w/v.
 5. The composition ofclaim 3, wherein the blood product is a modified cryo-poor plasma. 6.The composition of claim 4, wherein the blood product is a modifiedcryo-poor plasma.